Tight junctions have three critically important functions in both epithelia and endothelia. Tight junctions form the paracellular barrier that separates body compartments, they act as a fence that maintains apical-basolateral polarity of the cell, and they have paracellular pores that allow selective permeation of ions and small molecules across the cell monolayer. Three integral membrane proteins are thought to play a central role in these functions of the tight junction and hence are of high biological interest: claudin (a multigene family with 24 members), occludin, and tricellulin. The long-term goal of this proposal is to solve the structure of each of these proteins at atomic resolution and to use structure-guided mutagenesis to elucidate the structural mechanisms underlying their unique functions. The PI has already assembled a multidisciplinary team of investigators from Los Angeles, Pittsburgh, Boston, and Berlin to tackle the challenging problem of unraveling the function of claudins. This team has unique expertise in assaying paracellular pore function by electrophysiological methods, visualizing tight junction structure by freeze fracture electron microscopy, and hybrid molecular/Brownian dynamics modeling of pore function. The PI previously established a collaboration with the laboratory of Dr. Robert Stroud to express claudin proteins with the goal of crystallization, and has made significant progress. Thus, the applicants constitute a team that is uniquely poised to partner with a PSI Biology Network Center for Membrane Protein Structure Determination to tackle the structure determination and functional characterization of tight junction membrane proteins. Aim 1. Solve the structure of claudin by X-ray crystallography Aim 2. Determine the molecular basis of the pore functions of claudin Aim 3. Determine the structural basis of the barrier and fence functions of claudin Aim 4. Structure-function comparisons of tight junction membrane proteins PUBLIC HEALTH RELEVANCE: Tight junctions are junctions between cells that are needed for epithelia (e.g. skin, kidney, intestine) to act as barriers. This proposal would determine the structure of proteins in the membrane of the tight junction and use this information to elucidate the mechanism by which they function. This work has widespread significance for understanding diseases due to epithelial barrier dysfunction.